Staphylococcus aureus is a gram positive, ubiquitous bacterial species, with the predominant reservoir in nature being humans. The increased use of implanted medical devices such as intramedullary rods, screws, plates, and artificial joints has provided a physiological niche for microbes to cause infections. While a number of bacterial species may cause microbial fouling of indwelling medical devices, S. aureus causes a majority of these infections, producing high chronicity, morbidity, and mortality. One of the important mechanisms by which S. aureus is able to cause persistent infections on indwelling medical devices is through colonizing and synthesizing a "slime" layer, termed the glycocalyx or biofilm. This layer prevents infection resolution by antimicrobial agents and host phagocytic cells. Once an implant is colonized and chronic infection ensues, the standard treatment option is implant removal. This proposal seeks to identify S. aureus gene products with upregulated production in biofilms using two dimensional (2D) gel electrophoresis. Selected up-regulated proteins will then be evaluated, first, for their ability to be recognized by the host immune system during an in vivo biofilm infection, and second, for their protective efficacy in preventing infections in an implant-associated infection model in rabbits. The data generated here may contribute to the eventual development of a vaccine against S. aureus biofilms infections in humans. In addition, this proposal will also contribute to a more complete understanding of the bacterial factors involved in S. aureus biofilm formation and maturation. This understanding will enable one to create novel materials, surfaces, and/or disinfection strategies that resist or eliminate staphylococcal fouling and biofilm formation. Also, the proteome will be compared to transcriptome DMA microarray studies already completed in the Pi's laboratory in order to determine the global interrelation between gene expression and protein production for S. aureus biofilms grown under flow, thereby allowing staphylococci to be understood at a new level. Lastly, the results obtained in the evaluation of S. aureus biofilm formation may be used as a model for the biofilm formation by other closely related gram positive bacterial species, including Streptococcus spp., Listeria spp., Clostridium spp. and Bacillus spp.